Angiotensin-converting enzyme overexpression in mouse neutrophils prevents Alzheimer’s-like cognitive decline

Figures

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  Figure 1. Analysis of cognitive function in AD-NeuACE and AD mice. (A) The creation of AD-NeuACE mice was achieved by crossing NeuACE mice with 3xTg-AD mice. (B) ACE expression in neutrophils from AD and AD-NeuACE mice was examined using flow cytometry. The results are displayed as a histogram (left) and mean fluorescence intensity (MFI) (right). Data analysis was performed using a two-sided unpaired Student’s t-test, with data represented as means ± SEM (n = 3, ***p < 0.001). (C) The quantity of mice per genotype and age group (AD, AD-NeuACE, and WT) used in the behavior experiment is detailed. (D–G) Cognitive function in AD, AD-NeuACE, and WT mice (5–12 mice/group) was evaluated using the Barnes maze test. The data is represented as total latency (seconds) during the acquisition/training phase (Days 1–4) (D), memory retention (Day 7) (E), reversal phase (Days 8–9) (F), and memory test on Day 9 (G). (H–J) The Barnes maze test was also utilized to assess cognitive function in AD, AD-NeuACE, and WT mice (5–12 mice/group). The data is represented as primary latency (seconds) (H), total incorrect entries (errors) (I), and primary errors (J) during the acquisition/training phase (Days 1–4),…

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  Figure 2. Overexpression of ACE in neutrophils correlates with decreased Aβ levels. (A) Histological examination of Aβ in the cortex and hippocampus of AD, AD-NeuACE, and WT brains was conducted. The magnification used was ×100, and the scale bar represents 200 µm. The intensity of Aβ staining was analyzed using ImageJ software, and the quantitative data from all samples are displayed in the bar chart. Each dot on the graph signifies an individual sample. Group comparisons were analyzed using a one-way ANOVA with Bonferroni’s correction for multiple comparisons, and data are represented as means ± SEM. *p < 0.05, **p < 0.01, and ****p < 0.0001. (B) Representative immunofluorescence images showing colocalization of ACE1 (magenta), Ly6G+ neutrophils (red), and Aβ plaques (green) in hippocampal sections. (C, D) In-vitro phagocytic degradation analysis of fluorescent (FAM)-labeled β-Amyloid (FAM-Aβ). Blood neutrophils were incubated with FAM-Aβ for 30 min. After washing the neutrophils with FCM buffer, the FAM intensity of the neutrophils was assessed immediately (0h) or after a 2h incubation using flow cytometry (B) or by fluorescent microscope (C). Comparisons were analyzed using a two-sided…

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  Figure 3. Flow cytometry analysis of brain-infiltrating neutrophils and resident myeloid cells in AD and AD-NeuACE mice. (A) Quantification of neutrophil infiltration showing (left) absolute cell numbers (×104 CD45+) and (right) frequency as percentage of total CD45+ cells. (B) Amyloid-β uptake by brain neutrophils displayed as (left) fold change in Aβ mean fluorescence intensity (MFI), (middle) correlation between Aβ MFI and neutrophil frequency, and (right) representative flow cytometry histograms showing Aβ signal in AD (blue) versus AD-NeuACE (red) neutrophils compared to fluorescence-minus-one (FMO) control. (C) Expression of chemokine receptors CXCR2 (left) and CXCR4 (right) shown as MFI fold change. (D) Activation markers on brain neutrophils: CD80 MFI (left), CD86 MFI (middle), and MHC class II MFI (right). (E) ACE expression in brain myeloid cells: (left) representative flow cytometry plots showing gating strategy for macrophages (CD11b+CD45high) and microglia (CD11b+CD45low), (middle) ACE MFI in brain macrophages, and (right) ACE MFI in microglia, demonstrating no change in ACE expression in these populations in NeuACE mice. All data shown as mean ± SEM (n=2/3 per group).…